In vitro comparative activity of meropenem with 15 other antimicrobial agents against 1798 Pseudomonas aeruginosa isolates in a French multicenter study
INTRODUCTION
Pseudomonas aeruginosa is one of the most common pathogens involved in hospital-acquired infections. In recent years, a number of studies have reported an increased incidence of multiresistant strains [1–3] through the acquisition of plasmid-encoded β-lactamases [4], or aminoglycoside-modifying enzymes [5] and mutations. Mutational events are involved in the hyperproduction of the AmpC (class I) chromosomal β-lactamase that confers resistance to antipseudomonal penicillins and cephalosporins [6], the alteration of DNA gyrase that results in resistance to quinolones [7], the loss of the OprD2 porin in relation to imipenem resistance [8, 9], and the changes in the structure and energetics of the membrane that reduce drug accumulation by accelerating multidrug efflux [10] Because of this variety of resistance mechanisms, treatment of pseudomonal infections is difficult, and high morbidity and mortality among patients compromised by surgical wounds, burns, trauma and cancer are now observed [11].
Meropenem is a new carbapenem antibiotic that is more stable than imipenem to human renal dehydro-peptidase [12] and is more potent against many species, including P. aeruginosa [13]. We studied the activity of meropenem, in comparison with 15 other antimicrobial agents, against 1798 clinical isolates of P. aeruginosa collected from 10 French hospitals. The distribution of antimicrobial resistance and O serotypes was also examined according to hospital.
One thousand seven hundred and ninety-eight consecutive single clinical isolates of P. aeruginosa were collected over a period of 6 months (March to September 1996) from 10 hospital laboratories throughout France. Multiple isolates from the same patient were excluded if they belonged to the same serotype and had the same resistance pattern. Determination of the O serotype was made by slide agglutination with a set of four pools (OMA, OMC, OME, OMF) and 16 monovalent antisera numbered O1 to O16 (Sanofi Diagnostics Pasteur, Paris, France).
The susceptibility to antibiotics was determined by the disk diffusion method using the same lot of Mueller–Hinton agar and the following disks provided by Sanofi Diagnostics Pasteur: ticarcillin (75 mg), ticarcillin+clavulanate (75/10 mg), piperacillin (75 mg), piperacillin+tazobactam (75/10 mg), aztreonam (30 mg), ceftazidime (30 mg), cefepime (30 mg), cefsulodine (30 mg), imipenem (10 mg), meropenem (10 mg), gentamicin (15 mg), tobramycin (10 mg), netilmycin (30 mg), amikacin (30 mg), ciprofloxacin (5 mg), fosfomycin glucose 6-phosphate (50 mg). The zone sizes used to classify the strains as susceptible of resistant were defined by the CA of SFM [14].
When the isolate was resistant to ticarcillin, minimum inhibitory concentrations (MICs) of antibiotics were determined by an agar dilution method with Mueller–Hinton agar. Aliquots of 5X104 CPU per spot were inoculated on agar plates that contained two-fold serial dilutions of antibiotics. The MIC was the lowest concentration of antibiotic that completely inhibited visible growth after incubation for 18 h at 37°C.
The susceptibilities determined by the disk diffusion test for the 1798 strains of P. aeruginosa are reported in Table 1. Meropenem was the most potent agent, with a rate of susceptible isolates of 89.2%. For the other β-lactams, the rates of susceptible strains were as follows: ceftazidime 87.2%, imipenem 84.9%, piperacillin+tazobactam 84.7%, piperacillin 81.7%, aztreonam 80.6%, cefepime 77%, cefsulodin 75.9%, ticarcillin 65.8%, and ticarcillin+clavulanate 63.6%. There were also some differences in activity among the other antibiotics tested: thus, amikacin, tobramycin and ciprofloxacin displayed antibacterial activity by being active against 82%, 72.3% and 67.6% of isolates respectively, while gentamicin and fosfomycin were active against only 53.9% and 43% respectively.
| % of susceptible isolates for center | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | All centers | |
| Antibiotics | (n=196) | (n=186) | (n=121) | (n=192) | (n=206) | (n=180) | (n=188) | (n=157) | (n=190) | (n=182) | (n=1798) |
| Meropenem | 90 | 91 | 98 | 84 | 78 | 96 | 92 | 85 | 94 | 90 | 89.2 |
| Imipenem | 87 | 88 | 95 | 79 | 84 | 83 | 86 | 76 | 85 | 91 | 84.9 |
| Ticarcillin | 69 | 64 | 83 | 67 | 71 | 72 | 70 | 68 | 60 | 40 | 65.8 |
| Ticarcillin+clavulanate | 70 | 67 | 84 | 64 | 49 | 76 | 68 | 62 | 65 | 40 | 63.6 |
| Piperacillin | 87 | 83 | 94 | 75 | 75 | 85 | 85 | 72 | 83 | 82 | 81.7 |
| Piperacillin+tazobactam | 88 | 87 | 96 | 77 | 76 | 91 | 88 | 78 | 84 | 86 | 84.7 |
| Ceftazidime | 87 | 84 | 95 | 78 | 85 | 90 | 94 | 87 | 93 | 84 | 87.2 |
| Cefepime | 83 | 76 | 98 | 67 | 70 | 79 | 89 | ND | 75 | 63 | 77.0 |
| Cefsulodine | 84 | 68 | 93 | 71 | 64 | 86 | 85 | 54 | 80 | 78 | 75.9 |
| Aztreonam | 85 | 87 | 95 | 73 | 80 | 84 | 86 | 79 | 77 | 65 | 80.6 |
| Gentamicin | 57 | 51 | 72 | 45 | ND | 45 | 54 | 52 | 72 | 41 | 53.9 |
| Tobramycin | 81 | 81 | 75 | 58 | 71 | 84 | 71 | 62 | 83 | 54 | 72.3 |
| Netilmicin | 67 | 59 | 91 | 29 | 63 | 63 | 59 | 60 | 71 | 32 | 58.4 |
| Amikacin | 86 | 87 | 98 | 67 | 85 | 87 | 81 | 79 | 88 | 67 | 82.0 |
| Ciprofloxacin | 71 | 73 | 69 | 64 | 64 | 72 | 69 | 62 | 72 | 61 | 67.6 |
| Fosfomycin | 44 | 45 | 88 | 28 | 39 | 31 | 39 | 54 | 57 | 22 | 43.0 |
- ND, not defined.
The frequency of susceptible isolates varied according to the center. Isolates from center 10, 9, 4 and 2 were less susceptible to ticarcillin (40–47%), and those from centers 2, 4, 5, 8 and 10 less susceptible to cefsulodin (64–68%) or to cefepime (63–76%), than in other centers. Meropenem was the most effective agent against all isolates of P. aeruginosa collected in each center except for centers 5 and 10, in which the rate of isolates susceptible to meropenem was similar or slightly lower than that of imipenem.
The most common serotypes were O6 (16.9%), O11 (11.8%), O1 (10.7%), O12 (8.5%), O4 (6.6%) and O3 (6.3%), and 15% of isolates were non-typable. However, we noted that the range of serotype frequencies was particularly wide for serotypes O4 (0–23.1%), O11 (6.9–23.3%), and O12 (0.8–18.8%), as well as non-typable isolates (1.7–40.4%). Of the O12 strains, 39.4% were isolated from patients in intensive care units (data not shown)
The antibiotic susceptibilities of isolates according to serotypes are summarized in Table 2. The lowest susceptibility to all antibiotics except meropenem, ceftazidime and fosfomycin was found in the isolates belonging to serotype O12. By comparing the remaining serotypes and non-typable isolates, we found that the isolates belonging to serotype O4 were markedly less susceptible to ticarcillin alone or associated with clavulanate, tobramycin, netilmicin, ciprofloxacin and gentamicin, and that the isolates belonging to serotype O11 were frequently less susceptible to cephalosporins, imipenem and meropenem.
| % of susceptible isolates for serotype | |||||||
|---|---|---|---|---|---|---|---|
| O1 | O3 | O4 | O6 | O11 | O12 | NT | |
| Antibiotics | (n=193) | (n=113) | (n=119) | (n=304) | (n=212) | (n=153) | (n=270) |
| Meropenem | 96 | 95 | 94 | 94 | 86 | 70 | 89 |
| Imipenem | 94 | 94 | 92 | 91 | 86 | 41 | 84 |
| Ticarcillin | 79 | 80 | 51 | 75 | 63 | 14 | 65 |
| Ticarcillin+clavulanate | 74 | 78 | 52 | 70 | 59 | 14 | 66 |
| Piperacillin | 95 | 96 | 90 | 94 | 71 | 25 | 83 |
| Piperacillin+tazobactam | 95 | 97 | 92 | 96 | 80 | 29 | 85 |
| Ceftazidime | 94 | 95 | 86 | 93 | 78 | 72 | 84 |
| Cefepime | 90 | 89 | 60 | 87 | 68 | 32 | 74 |
| Cefsulodine | 91 | 88 | 84 | 88 | 71 | 25 | 74 |
| Aztreonam | 87 | 92 | 72 | 88 | 79 | 49 | 79 |
| Gentamicin | 66 | 63 | 24 | 69 | 37 | 21 | 53 |
| Tobramycin | 90 | 87 | 33 | 87 | 63 | 22 | 76 |
| Netilmicin | 72 | 71 | 33 | 72 | 45 | 18 | 58 |
| Amikacin | 88 | 90 | 77 | 96 | 79 | 37 | 81 |
| Ciprofloxacin | 87 | 82 | 33 | 86 | 60 | 13 | 70 |
| Fosfomycin | 44 | 36 | 45 | 54 | 30 | 65 | 40 |
- NT, non-typable strains.
The MICs obtained for isolates of P. aeruginosa that were either resistant to ticarcillin and susceptible to ceftazidime (group 1: 148 strains) or resistant to ticarcillin and ceftazidime (group 2: 156 strains) are summarized in Table 3. Most of the strains of group 1 were resistant to cefepime, piperacillin, ciprofloxacin and amikacin, with rates of susceptible strains of 11.5%, 15.5%, 23% and 34.5% respectively. Meropenem and imipenem were the most active antibiotics; nevertheless, the MIC50 of meropenem was lower than that of imipenem. In group 2, no strain was susceptible to cefepime, and few remained susceptible to amikacin and ciprofloxacin (26.9% and 17.3% respectively). According to MICs obtained for these resistant strains, about 60% of strains were susceptible to imipenem or meropenem. Finally, meropenem was active against 28.3% of imipenem-resistant strains.
| Group 1 n = 148 | Group 2 n=156 | |||||||
|---|---|---|---|---|---|---|---|---|
| Antibiotics | MIC50 | MIC90 | MIC range | % of susceptible isolates | MIC50 | MIC90 | MIC range | % of isolates susceptible |
| Meropenem | 1 | 8 | 0.128–32 | 83.1 | 2 | 32 | 0.128–64 | 60.3 |
| Imipenem | 4 | 16 | 0.25–64 | 79.7 | 4 | 32 | 0.5–128 | 62.8 |
| Ticarcillin | 1024 | 4096 | 128 to > 4096 | 0 | 512 | 2048 | 128 to > 4096 | 0 |
| Piperacillin | 256 | 1024 | 1 to > 1024 | 15.5 | 256 | 1024 | 16 to > 1024 | 4.5 |
| Cefepime | 16 | 32 | 1–64 | 11.5 | 16 | 64 | 8–256 | 0 |
| Ceftazidime | 2 | 4 | 0.5–4 | 100 | 16 | 64 | 8–512 | 0 |
| Amikacin | 16 | 64 | 0.128–256 | 34.5 | 16 | 256 | 1–512 | 26.9 |
| Ciprofloxacin | 32 | 64 | 0.032 to >64 | 23 | 32 | 64 | 0.064 to > 64 | 17.3 |
| Fosfomycin | 32 | 256 | 1–512 | 52 | 64 | 512 | 1 to >512 | 41 |
The high incidence of multiresistant P. aeruginosa isolates in hospital patients demands the development of new antibiotics. Most of the resistant strains were isolated from intensive care unit patients, where these antibiotics are widely used. Alternative drugs will soon be needed. The present study compared the in vitro activity of meropenem with that of 15 other antibiotics against 1798 P. aeruginosa strains collected from 10 different French centers. The results of this study showed that meropenem had the best activity, with a rate of susceptible strains of 89.2%. A similar finding was noted in other epidemiologic studies, with rates of susceptible strains of 90–95.8% [15, 16].
Susceptibility differences were noted among the 10 centers participating in the present collaborative study. Local variations of antibiotic pressure, but also variations in the incidence of some serotype O strains, might be responsible. Thus, most resistant strains belonged to serotype O12, and the centers with the highest rates of resistant strains were correlated with a high incidence of serotype O12. Thus, most strains of serotype O12 were resistant to aminoglycosides and β-lactam antibiotics as described in other countries [17]. Most O12 isolates displayed a particular profile consistent with the hypothesis of only some multi-resistant strains spreading through Europe [17, 18]. The greatest frequency of this serotype in intensive care units might explain its selection by drug pressure. Meropenem, ceftazidime and fosfomycin were the best agents against strains of this serotype. The distribution of serotypes O1, O3 and O6 which included the most susceptible strains also varied according to center: they were more frequent in centers with low prevalences of serotype O12 than in the others. Their susceptibilities to antibiotics were similar to those described in other European epidemiologic studies [19, 20].
Meropenem appeared to be active in vitro against many clinical isolates of P. aeruginosa that are resistant to ticarcillin and/or to ceftazidime. This observation is in agreement with the findings of other studies that reported meropenem susceptibility in multiresistant isolates from patients [21], and with the greater stability of meropenem against Pseudomonas β-lactamases [3, 5]. Of the two carbapenems, meropenem showed the best intrinsic activity, with lower MIC50S against these two populations of resistant strains. Furthermore, it was effective against 28.3% of imipenem-resistant isolates. Similar meropenem activity against imipenem-resistant strains was described by Garcia-Rodriguez et al [15], with 17% of imipenem-resistant strains being susceptible to meropenem. As described by Edwards et al [12], our strains, which remained susceptible to meropenem (MICs <4 mg), were only moderately imipenem-resistant strains with MICs between 8 and 32 mg/L, while highly imipenem-resistant strains (MIC≥64 mg/L) were invariably meropenem resistant. The primary mechanism in carbapenem resistance of P. aeruginosa is directly related to the absence or diminished expression of porin OprD2, a selective permeation pathway for carbapenems, resulting in reduced uptake of antimicrobial agents [8]. The susceptibility to meropenem of strains showing a low level of resistance to imipenem is possible because of another pathway for the translocation of meropenem across the outer membrane [22].
Masuda et al [23] described meropenem-resistant P. aeruginosa isolates which are susceptible to imipenem. This resistance was associated with overproduction of an outer-membrane protein presumably similar to OprM, an efflux pump implicated in intrinsic resistance [10]. It was associated with cross-resistance to cephems and quinolones. Only 0.7% of strains resistant to meropenem but susceptible to imipenem were found in our study.
In conclusion, meropenem is a promising carbapenem with antimicrobial activity comparable or superior to that of imipenem against P. aeruginosa. It should be considered as an effective agent in therapy for P. aeruginosa infections, with good activity against strains harboring resistance to β-lactam antibiotics. However, as with other antibiotics, we have to be careful of selection pressure encouraging the development of resistant strains.
